Allosteric activation of protein phosphatase 2C by D-chiro-inositol-galactosamine, a putative mediator mimetic of insulin action.

Insulin-stimulated glucose disposal in skeletal muscle proceeds predominantly through a nonoxidative pathway with glycogen synthase as a rate-limiting enzyme, yet the mechanisms for insulin activation of glycogen synthase are not understood despite years of investigation. Isolation of putative insulin second messengers from beef liver yielded a pseudo-disaccharide consisting of pinitol (3-O-methyl-d-chiro-inositol) beta-1,4 linked to galactosamine chelated with Mn(2+) (called INS2). Here we show that chemically synthesized INS2 has biological activity that significantly enhances insulin reduction of hyperglycemia in streptozotocin diabetic rats. We used computer modeling to dock INS2 onto the known three-dimensional crystal structure of protein phosphatase 2C (PP2C). Modeling and FlexX/CScore energy minimization predicted a unique favorable site on PP2C for INS2 in a surface cleft adjacent to the catalytic center. Binding of INS2 is predicted to involve formation of multiple H-bonds, including one with residue Asp163. Wild-type PP2C activity assayed with a phosphopeptide substrate was potently stimulated in a dose-dependent manner by INS2. In contrast, the D163A mutant of PP2C was not activated by INS2. The D163A mutant and wild-type PP2C in the absence of INS2 had the same Mn(2+)-dependent phosphatase activity with p-nitrophenyl phosphate as a substrate, showing that this mutation did not disrupt the catalytic site. We propose that INS2 allosterically activates PP2C, fulfilling the role of a putative mediator mimetic of insulin signaling to promote protein dephosphorylation and metabolic responses.

Oxidant regulation of gene expression and neural tube development: Insights gained from diabetic pregnancy on molecular causes of neural tube defects.

AIMS/HYPOTHESIS: Maternal diabetes increases oxidative stress in embryos. Maternal diabetes also inhibits expression of embryonic genes, most notably, Pax-3, which is required for neural tube closure. Here we tested the hypothesis that oxidative stress inhibits expression of Pax-3, thereby providing a molecular basis for neural tube defects induced by diabetic pregnancy. METHODS: Maternal diabetes-induced oxidative stress was blocked with alpha-tocopherol (vitamin E), and oxidative stress was induced with the complex III electron transport inhibitor, antimycin A, using pregnant diabetic or non-diabetic mice, primary cultures of neurulating mouse embryo tissues, or differentiating P19 embryonal carcinoma cells. Pax-3 expression was assayed by quantitative RT-PCR, and neural tube defects were scored by visual inspection. Oxidation-induced DNA fragmentation in P19 cells was assayed by electrophoretic analysis. RESULTS: Maternal diabetes inhibited Pax-3 expression and increased neural tube defects, and alpha-tocopherol blocked these effects. In addition, induction of oxidative stress with antimycin A inhibited Pax-3 expression and increased neural tube defects. In cultured embryo tissues, high glucose-inhibited Pax-3 expression, and this effect was blocked by alpha-tocopherol and GSH-ethyl ester, and Pax-3 expression was inhibited by culture with antimycin A. In differentiating P19 cells, antimycin A inhibited Pax-3 induction but did not induce DNA strand breaks. CONCLUSION/INTERPRETATION: Oxidative stress inhibits expression of Pax-3, a gene that is essential for neural tube closure. Impaired expression of essential developmental control genes could be the central mechanism by which neural tube defects occur during diabetic pregnancy, as well as other sources of oxidative stress.

Evidence that elevated glucose causes altered gene expression, apoptosis, and neural tube defects in a mouse model of diabetic pregnancy.

Congenital malformations, including neural tube defects (NTDs), are significantly increased in the offspring of diabetic mothers. We previously reported that in the embryos of a mouse model of diabetic pregnancy, NTDs are associated with reduced expression of the gene Pax-3, which encodes a transcription factor that regulates neural tube development, and that reduced expression of Pax-3 leads to neuroepithelial apoptosis. In this study, we used three approaches to test whether glucose alone could be responsible for these adverse effects of diabetes on embryonic development. First, primary culture of embryo tissue in medium containing 15 mmol/l glucose inhibited Pax-3 expression compared with culture in medium containing 5 mmol/l glucose. Second, inducing hyperglycemia in pregnant mice by subcutaneous glucose administration significantly inhibited Pax-3 expression (P < 0.05), as demonstrated by quantitative reverse transcription-polymerase chain reaction assay of Pax-3 mRNA, and also increased neural tube apoptosis (P < 0.05). NTDs were significantly increased in glucose-injected pregnancies when blood glucose levels were >250 mg/dl (P < 0.002) but not in moderately hyperglycemic pregnancies (150-250 mg/dl, P = 0.37). Third, phlorizin administration to pregnant diabetic mice reduced blood glucose levels and the rate of NTDs. As seen with glucose-injected pregnancies, the rate of NTDs in phlorizin-treated diabetic pregnancies was related to the severity of hyperglycemia, since NTDs were significantly increased in severely hyperglycemic (>250 mg/dl) diabetic pregnancies (P < 0.001) but not in moderately hyperglycemic pregnancies (150-250 mg/dl, P = 0.35). These two findings, that elevated glucose alone can cause the changes in Pax-3 expression observed during diabetic pregnancy and that the NTD rate rises with significant increases in blood glucose levels, suggest that congenital malformations associated with diabetic pregnancy are caused by disruption of regulatory gene expression in the embryo in response to elevated glucose.

Diagnosis of hypoglycaemia: effects of blood sample handling and evaluation of a glucose photometer in the low glucose range.

Hypoglycaemia is a dangerous condition. Rapid and reliable blood glucose measurements are necessary for the initiation of treatment to reduce the risk of neurological sequelae. The aim of this study was to compare a bedside glucose photometer (HemoCue) with three methods of handling blood glucose measurements in a routine chemistry laboratory and to estimate the reliability of glucose measurements in the low glucose range during controlled hypoglycaemia. Nine children underwent an arginine-insulin tolerance test as part of a growth hormone deficiency investigation. Only blood samples below 4.0 mmol l-1 were included (n = 35). Significant (0.3-1.0 mmol l-1) differences in blood glucose measurements were found, depending on the handling of the blood sample. The differences seem primarily to be due to glycolysis which occurred in spite of the addition of the glycolysis inhibitor NaF to the blood samples. Immediate centrifugation and analysis of the supernatant or immediate analysis with the HemoCue results in higher, and presumably more correct, values than routine procedures and permits a more accurate diagnosis of hypoglycaemia.

Routine measurements of umbilical artery lactate levels in the prediction of perinatal outcome.

OBJECTIVE: Our purpose was to compare lactate levels with acid-base balance in the umbilical artery with respect to the prediction of pregnancy outcome. STUDY DESIGN: A prospective study of 4045 cord samples was performed. Lactate was measured with a new method that requires 5 microliters of blood and provides the result within 1 minute. RESULTS: The umbilical artery lactate concentrations were significantly elevated in instrumental deliveries (2.65 +/- 1.2 mmol/L) and in emergency cesarean sections (2.44 +/- 1.7 mmol/L) compared with spontaneous vaginal delivery (1.87 +/- 0.94 mmol/L) (p < 0.001, p < 0.001). Lactate correlated significantly to fetal pH, hemoglobin, base deficit, PCO2, and HCO3-. Lactate was comparable to pH and base deficit in sensitivity, specificity, and positive and negative predictive values in relation to morbidity and mortality. CONCLUSION: Umbilical artery lactate concentration and acid-base balance predicted perinatal outcomes with similar efficacies; however, its simplicity makes lactate analysis an interesting alternative in obstetric care.

Metabolic adaptation in IUGR neonates determined with microdialysis--a pilot study.

Subcutaneous microdialysis was used to monitor the immediate metabolic changes with respect to glucose, glycerol and lactate in the extracellular space of adipose tissue among five small-for-gestational-age (SGA) and two appropriate-for-gestational-age (AGA) newborns. There was a good correlation between glucose levels in blood and dialysate (r = 0.97, n = 14). The infants showed rapid rises and falls in dialysate glucose levels that are not seen among older children and adults. The levels of lactate were higher than those reported in blood. Lactate may serve as an alternative source of energy for the neonate. Microdialysis is of potential value in increasing our understanding of metabolic events. It provides a safe on-line means of making continuous measurements in these fragile patients in order to detect periods of hypoglycaemia, at least in infants with a birth weight > 1000 g.